Abstract The Notch signaling pathway directs cell fate decisions in development of nearly every tissue, plays key roles in numerous diseases, and represents a major drug target. The pathway uses multiple ligands and receptors that interact with one another in a promiscuous fashion, as well as Fringe glycosyltransferases that modulate those interactions. Recent work from our lab has revealed that these interactions comprise a communication ?code? in which different ligands activate different target programs, even through the same receptors. The code appears to function through a dynamic encoding mechanism, in which different ligands activate Notch receptors in either a pulsatile or sustained fashion. These different dynamics in turn selectively activate distinct target programs. Current understanding of the code is limited to just two ligands and one receptor. By elucidating the full code, covering all ligand-receptor-Fringe combinations, we will enable quantitative prediction of signaling interactions between cells with arbitrary Notch component expression profiles across different developmental and disease contexts. To decipher this code and its functional roles, we will combine cell line engineering, quantitative single-cell time-lapse imaging, direct control of Notch dynamics using mutant receptors and pharmacological perturbations, and analysis of Notch dynamics in neural stem cells and chick embryos. In ?Aim 1??, we will map dynamic signaling modes (pulsatile or sustained) across a full matrix of Notch receptor, ligand, and Fringe protein combinations. In ?Aim 2??, we will analyze dynamic signal decoding by determining whether receptor intracellular domain composition affects target gene expression, or whether target program specificity depends only on the strength and dynamics of signaling. Finally, we will analyze the function of the Notch dynamic code in chick spinal cord development (?Aim 3??). Specifically, we will use a fluorescent reporter exclusively activated by Notch, together with a new tissue slice preparation that enables imaging of individual living cells during spinal cord development, to link Notch dynamics to cell fate determination. Together, these results will reveal the structure and function of the dynamic code underlying Notch signaling, and show how it operates in a central vertebrate developmental process.